EP3479158B1 - Arrangement for microscopy and for correction of aberrations - Google Patents
Arrangement for microscopy and for correction of aberrations Download PDFInfo
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- EP3479158B1 EP3479158B1 EP17734078.3A EP17734078A EP3479158B1 EP 3479158 B1 EP3479158 B1 EP 3479158B1 EP 17734078 A EP17734078 A EP 17734078A EP 3479158 B1 EP3479158 B1 EP 3479158B1
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- 238000012937 correction Methods 0.000 title claims description 88
- 230000004075 alteration Effects 0.000 title claims description 46
- 238000000386 microscopy Methods 0.000 title description 7
- 238000005286 illumination Methods 0.000 claims description 89
- 238000001514 detection method Methods 0.000 claims description 87
- 230000003044 adaptive effect Effects 0.000 claims description 46
- 230000003287 optical effect Effects 0.000 claims description 42
- 230000005855 radiation Effects 0.000 claims description 36
- 210000001747 pupil Anatomy 0.000 claims description 19
- 230000005499 meniscus Effects 0.000 claims description 14
- 238000007654 immersion Methods 0.000 claims description 8
- 230000003068 static effect Effects 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 4
- 201000009310 astigmatism Diseases 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims 2
- 239000006059 cover glass Substances 0.000 description 15
- 239000008186 active pharmaceutical agent Substances 0.000 description 8
- 238000007493 shaping process Methods 0.000 description 5
- 230000001419 dependent effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000010363 phase shift Effects 0.000 description 1
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- 230000000717 retained effect Effects 0.000 description 1
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Classifications
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
- G02B21/04—Objectives involving mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
Definitions
- the invention relates to a microscope according to the preamble of independent claim 1.
- One of the main applications of light sheet microscopy is in the imaging of medium-sized samples, for example organisms, with a size of a few 100 ⁇ m down to a few millimeters.
- these samples are embedded in agarose and placed in a glass capillary.
- the glass capillary is placed in a water-filled sample chamber. The agarose with the sample is squeezed out of the capillary a little and illuminated with a light sheet.
- a structure of a microscope 0 for light sheet microscopy comprises an illumination lens 2 with a first optical axis A1 and a detection lens 3 with a second optical axis A2 (also referred to as SPIM lenses ), which are each directed at an angle ⁇ 1 or a2 of 45° to a sample plane 4 and at a right angle to one another from above onto the sample plane 4 (see Fig. 1a ).
- a sample 5 arranged in the sample plane 4, which is also used as a reference plane, in a sample area provided for this purpose is located, for example, on the bottom of a sample holder 7 designed as a Petri dish.
- the sample holder 7 is filled with a medium 8, for example water, and the Both SPIM objectives 2, 3 are immersed in the medium 8 (not shown) during the application of light sheet microscopy.
- the sample plane 4 extends in a plane XY spanned by the X-axis X and the Y-axis Y of a Cartesian coordinate system.
- the first optical axis A1 and the second optical axis A2 run in a plane YZ spanned by the Y axis Y and the Z axis Z of the Cartesian coordinate system.
- the Fig. 1b shows schematically a microscope 0 with an inverse arrangement of illumination lens 2 and detection lens 3 according to the prior art, in which the illumination lens 2 and the detection lens 3 are arranged below the sample plane 4 .
- the angles ⁇ 1 and ⁇ 2 are again each 45°.
- the aberrations caused by the sample holder 7, which is inclined relative to the optical axes A1 and A2 and is in the form of a cover glass, must be corrected by special optical elements.
- the sample 5 arranged in the sample plane 4 is illuminated through the bottom of the sample holder 7 and an excited fluorescence of the sample 5 is detected.
- Sample holder 7 such as. B. multiwell plates, petri dishes and / or slides and contamination of the samples 5, especially in high-throughput screening, can be avoided.
- Alvarez plates of an Alvarez manipulator 12 ( Fig. 1b , the Alvarez plates are designated for simplification) are arranged like these, for example, from the US3305294A are known.
- the Alvarez plates are designed in such a way that they correct aberrations occurring precisely at a fixed angle between the sample holder 7, for example a cover glass, and the optical axis A1, A2 of the respective objective 2, 3. Even a small deviation in the angle (e.g. ⁇ 0.1°) will result in unwanted aberrations that lead to reduced imaging quality. Therefore, before starting an experiment, the coverslip, for example, must be aligned so that the angular deviation is within the permissible tolerances. It is also helpful if, in addition to the angle, the distance between the objective 2, 3 or any additional lens that may be present and the cover glass can be adjusted, so that the sample 5 or its area to be imaged lies in the image plane BE of the detection objective 3.
- a virtual relay is known, which is used to correct errors that occur when the beams pass through a slide at an angle. Since the virtual relay has a high numerical aperture > 1.2, slight deviations within the optical system, which can vary from experiment to experiment, can result in pronounced imaging errors. The deviations can be based, among other things, on the variance of the cover glass thickness, temperature changes, differences in the refractive index, tilting of the cover glass or wedge errors in the cover glass.
- shift lenses can be provided in a lens to correct aberrations.
- Another, also in the DE 10 2013 107 297 A1 The possibility described consists in the arrangement of adaptive mirrors or spatial light modulators for light (spatial light modulator; SLM) in the frequency domain (e.g. in a pupil) outside the lens.
- SLM spatial light modulator
- the invention is based on the object of proposing improved options for correcting aberrations compared to the prior art, which in particular are due to oblique passages of illumination radiation and detection radiation through optically refracting layers appear.
- improved microscopes and here in particular light sheet microscopes, are to be proposed.
- the task is solved using a microscope.
- the microscope comprises illumination optics with an illumination objective for illuminating a sample located on a sample carrier in a sample area via an illumination beam path, the optical axis of the illumination objective lying in a plane that is one of Includes zero different angles (illumination angles).
- the illumination by means of the illumination lens takes place in said plane.
- the optical axis of the detection lens encloses a non-zero angle (detection angle) with the normal of the reference plane.
- the detection objective includes a detection correction element that is arranged in the beam path or can be introduced into it and/or the illumination objective includes an illumination correction element that is arranged in the beam path or can be introduced into this.
- a meniscus lens is present between the sample carrier and the two objectives, which is arranged both in the illumination beam path and in the detection beam path.
- the meniscus lens is designed to correct aberrations that occur due to the passage of radiation to be detected, in particular light, or radiation for illuminating the sample through media of different refractive power.
- the correction element or the correction elements is or are designed to correct remaining aberrations.
- the sample In the sample plane, which is also referred to as the reference plane, the sample is or can be arranged in a designated area, the sample area.
- the lighting is flat. To simplify the description, reference is also made below to a correction element or correction elements if the description relates both to an illumination correction element and to a detection correction element, or to both.
- Remaining aberrations can be those (residual) aberrations that result from an incomplete correction of the aberrations due to the oblique passage of the radiation, be it illumination radiation and/or detection radiation.
- remaining aberrations are errors, for example due to a variance in the cover glass thickness, temperature changes, differences in the refractive index of layers penetrated by radiation, tilting of the cover glass or wedge errors in the cover glass. These remaining aberrations are corrected or at least reduced.
- the bottom of a sample vessel or a slide made of a material other than glass can be equated with a cover glass.
- the microscope can have a separating layer system with at least one layer made of a specified material with a specified thickness.
- the at least one layer for example a cover glass, separates a medium in which the sample is located from the illumination objective and the detection objective.
- the separating layer system stands, with a base surface aligned parallel to the reference plane, at least in the area for the illumination lens and the Detection objective for illumination or detection accessible area with the medium and / or with an immersion medium in contact. The medium and the immersion medium are separated from each other by the separating layer system.
- the aberrations and the remaining aberrations can be reduced for a predetermined range of illumination angles or detection angles and/or for a predetermined range of the thickness of the at least one layer of the separating layer system.
- a meniscus lens is a lens that has two lens surfaces curved in the same direction.
- the two lens surfaces advantageously have the same center point.
- the two lens surfaces of the meniscus lens can be in different media, for example immersion media and/or air, each with a different refractive index.
- the meniscus lens has the advantage over the virtual relay known from the prior art and over previously known free-form correction lenses that it can be produced more simply and cost-effectively, since no free-form surfaces have to be produced in a complex manner.
- the meniscus lens can be held stationary.
- the focusing is done by moving the sample together with the sample carrier or by moving the lenses along their optical axis.
- the meniscus lens is used to correct or correct errors that occur when the illumination radiation and/or detection radiation transitions between two media or layers of different refractive index. However, aberrations due to the oblique passage are not corrected. These remaining aberrations can be corrected outside and/or inside the lens by means of the illumination correction element and/or by means of the detection correction element (correction elements).
- a radiation used for illumination is formed into a light sheet and directed into the sample area.
- the light sheet is generated by means of the illumination radiation in the sample area, for example by moving a bundle of rays of the illumination radiation in the plane (dynamic light sheet).
- the optical axis of the illumination lens and the light sheet lie in a plane that encloses an illumination angle other than zero with the normal of the reference plane.
- the optical correction element is arranged in a pupil of the detection objective and/or the illumination objective.
- Illumination lens and detection lens are also referred to below as lenses for the sake of simplicity.
- An arrangement of the optical correction element in a pupil or as close as possible to the pupil advantageously avoids undesired field-dependent effects.
- the correction element In the pupil, the correction element has the same effect for all field points. The greater the distance between the pupil and the respective optical correction element, the more field dependencies come into play, so that the undesired field-dependent effects are more pronounced as the distance increases.
- the optical correction element is arranged close to the pupil if it is within the depth of focus, for example, of the tube lens of the detection objective or of the illumination objective.
- Both the optical illumination correction element and the optical detection correction element can be embodied as static correction elements or as adaptive correction elements.
- Static correction elements are, for example, at least one phase plate or one free-form lens.
- the free-form lens does not necessarily have to be placed in the pupil and can be the front lens of the respective objective, for example.
- a static correction element such as the phase plate corrects static components of the aberrations.
- adaptive correction elements can be accommodated in the illumination and detection beam path of the arrangement. Dynamic or variable portions of the aberrations can be corrected by means of at least one adaptive correction element, with the adaptive correction element being or being arranged inside or outside the lens or lenses.
- An adaptive correction element is adjustable and adaptive in terms of its corrective effect. Dynamic corrections of the aberrations, in particular the remaining aberrations, are thus advantageously made possible.
- Static and adaptive correction elements can be combined in a microscope according to the invention.
- the static correction element is a phase plate for correcting static aberrations
- an adaptive correction element is arranged in the illumination and/or detection beam path.
- an adaptive correction element is assigned to each objective or if each of the objectives has an adaptive correction element, one of the adaptive correction elements can be present inside and the other adaptive correction element can be present outside of the respective objective.
- An adaptive correction element is expediently arranged in a pupil plane of the microscope in such a way that the effective opening of the adaptive correction element and the size of the variable adaptive correction element or the variable adaptive correction elements fit well with the size of the pupil in the pupil plane and desired wavefront deformations are set for the aberrations to be compensated and/or to supply a required phase shift for the aberrations to be compensated.
- this adaptation and the accessibility of the adaptive correction element are achieved by a pupil relay optics, which images the objective pupil of the illumination or detection objective onto the adaptive correction element.
- Sufficiently small adaptive correction elements can also be arranged directly in or immediately after the lens.
- Adaptive correction elements are, for example, adaptive mirrors or at least one spatial light modulator (SLM).
- the SLM can be designed as a reflective SLM or as a transmissive SLM.
- the adaptive correction element is an Alvarez manipulator, at least one adaptive mirror, at least one tilting lens, at least one shifting lens, at least one deformable optical lens or a combination thereof.
- the adaptive correction element is a spatial modulator for light while in the beam path of one of the lenses, in particular in the
- Detection beam path a cylindrical lens for partial compensation of aberrations that occur is present.
- the adaptive correction element prefferably be an adaptive mirror and for a cylindrical lens to be present in the beam path of one of the lenses, in particular in the detection beam path, for partial compensation of astigmatism that occurs.
- the pupil of the lens is imaged onto the adaptive mirror by means of a telescope.
- the adaptive mirror is deformed in such a way that it corrects and reduces the aberrations that occur.
- An almost or completely aberration-free image can be generated on the camera sensor by means of a further optical lens arranged in the detection beam path.
- the adaptive correction element is implemented by a reflecting SLM
- the pupil of the lens is imaged onto the SLM using a telescope.
- a phase pattern is displayed on the SLM, the effect of which is to correct and reduce the aberrations that occur.
- an almost or completely aberration-free image can be generated on the camera sensor by means of a further optical lens arranged in the detection beam path.
- the SLM is combined with a cylindrical lens.
- the cylindrical lens is z. B. used in the pupil of the lens to perform a partial correction of the aberrations that occur.
- the pupil of the lens is imaged onto a reflecting SLM using a telescope.
- a phase pattern is displayed on the SLM, the optical effect of which corrects and reduces the remaining residual aberrations.
- a third lens creates an almost or completely aberration-free image on the camera sensor.
- the adaptive correction elements can be arranged in the illumination and/or in the detection beam path.
- the aberrations can also be corrected within the lens.
- an additional pupil is created in the lens, for example, and the adaptive correction element is placed in its place in order to correct the aberrations that occur.
- a cylindrical lens can also be used here in order to carry out a partial correction of the aberrations.
- the invention is particularly suitable in the form of an inverted light sheet microscope with oblique passage of the illumination and detection radiation through a sample holder, for example in the form of a cover glass or an optically transparent layer such as the bottom of a Petri dish.
- the essential elements of an arrangement according to the invention for microscopy, in particular for light sheet microscopy, in addition to the illumination objective 2, which is aligned obliquely to the sample or reference plane 4, and the detection objective 3, which is also aligned obliquely to the reference plane 4, are a shared meniscus lens 10 ( 2 ) and at least one illumination correction element 2KE in the illumination objective 2 and/or at least one detection correction element 3KE in the detection objective 3.
- the angles ⁇ 1 and ⁇ 2 between a normal B perpendicular to the reference plane 4 and the first optical axis A1 or the second optical axis A2 are each 45°.
- Two Alvarez plates of an Alvarez manipulator 12 are arranged in the beam path of the illumination radiation BS and in the beam path of the detection radiation DS as adaptive correction elements 2KE, 3KE.
- the correction elements 2KE, 3KE are used to correct aberrations that can occur as a result of the oblique passage of the illumination radiation BS through the bottom of the sample holder 7 .
- the meniscus lens 10 supports the transition of the illumination radiation BS from air into an immersion medium 18 and into the medium 8 as well as the transition of the detection radiation DS from the medium 8 into the immersion medium 18 and into the air.
- the sample holder 7 is held on the sample table 11 .
- the sample table 11 itself can be adjusted in a controlled manner in an X-Y plane spanned by the X-axis X and the Y-axis Y by means of drives that are not shown in detail.
- the illumination lens 2 and the detection lens 3 can each be adjusted in a controlled manner along the first optical axis A1 or along the second optical axis A2 by means of a lens drive 14, which is designed here as a piezo drive.
- the illumination radiation BS is provided by a laser module 15 and shaped by a beam shaping unit 16 .
- the beam shaping unit 16 is, for example, an optical system by means of which the provided illumination radiation BS is shaped, for example collimated.
- a scanner 17 Downstream of the beam shaping unit 16 there is a scanner 17, by means of which the shaped illumination radiation BS can be deflected in a controlled manner in two directions (X-Y scanner).
- the illumination objective 2 is arranged on the first optical axis A1.
- the illumination radiation BS deflected by the scanner 17 reaches the illumination objective 2 and is shaped and/or focused by it.
- the light sheet 6 is generated by a corresponding deflection of the illumination radiation BS by means of the scanner 17 in a sample area in which the sample 5 is located.
- the detection radiation DS coming from the sample 5 and from the sample area is directed along the second optical axis A2 onto a detector 19 and can be detected by it.
- a control unit 13 is provided for controlling the sample table 11, the lens drives 14, the correction elements 2KE, 3KE, the laser module 15, the beam shaping 16, the scanner 17 and/or the detector 19, which is connected to the elements to be controlled in a connection suitable for data transmission stands (shown only implicitly).
- control unit 13 is additionally configured to record, store and/or evaluate measured values. Further elements and units of the microscope 0 can be controlled by means of the control unit 13 and/or measured values can be obtained and evaluated from them.
- the first coordinate system is the coordinate system of the entire arrangement with an X-axis X, a Y-axis Y and a Z-axis Z.
- the sample holder 7, in particular its bottom is parallel to one through the X-axis X and the Y -Axis Y spanned X-Y plane aligned.
- the second coordinate system is the coordinate system of the detector 19 with the X-axis X, a y-axis y' and a z-axis z'.
- An image, for example, of an image from the image plane BE on the detector 19 has the coordinates X and y'.
- the X-axis X is orthogonal to the drawing plane of the figures.
- the two other axes Y and y′ or Z and z′ can be converted into one another by rotating around the X axis.
- correction elements 2KE, 3KE are mounted displaceably in the illumination lens 2 and/or the detection lens 3 in order to adjust an aberration correction to the thickness by shifting the correction elements 12 relative to one another.
- the bottom of the sample holder 7 represents a separating layer system with at least one layer made of a predetermined material with a predetermined thickness, which separates a medium 8 in which the sample 5 is located from the illumination lens 2 and the detection lens 3 .
- the separating layer system is in contact with the immersion medium 18 at least in the area accessible to the illumination objective 2 and the detection objective 3 for the illumination or the detection with a base surface aligned parallel to the sample plane 4 .
- the illumination lens 2 in turn has an Alvarez manipulator 12 as
- Optical lenses 20 are present in the beam path of the detection radiation DS, by means of which the detection radiation DS is directed onto a
- Detection correction element 3KE is directed in the form of an SLM or an adaptive mirror.
- the detection radiation DS reflected by the detection correction element 3KE is directed onto a detector 19 and detected by it.
- the SLM is designed as a transmissive SLM.
- the 4 12 shows a third exemplary embodiment of the invention, a cylindrical lens 9 being arranged in front of the optical lenses 20 in the beam path of the detection radiation DS, which is used for partial compensation of astigmatism that occurs.
- the detection correction element 3KE is implemented in the form of an SLM or an adaptive mirror.
- FIG figure 5 Another exemplary embodiment of a microscope (0) according to the invention is shown in FIG figure 5 shown.
- the illumination objective 2 has no illumination correction element 2KE.
- Two detection correction elements 3KE are integrated in the detection objective 3 and designed as reflective(s) SLM and/or as adaptive(s) mirrors.
- FIG. 2 An embodiment of a lens 2.3 for use in a microscope 0 according to the invention (see Figures 2 to 5 ) is in the 6 shown schematically.
- a controllable correction element 2KE or 3KE embodied as an SLM, is arranged in the beam path. It is controlled by the control unit 13.
- sliding lenses can be provided which can be displaced radially with respect to the beam path in order to compensate for or at least reduce any remaining aberrations.
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Description
Die Erfindung betrifft ein Mikroskop gemäß dem Oberbegriff des unabhängigen Anspruchs 1. Eine der Hauptanwendungen der Lichtblattmikroskopie liegt in der Bildgebung mittelgroßer Proben, beispielsweise von Organismen, mit einer Größe von einigen 100 µm bis hin zu wenigen Millimetern. In der Regel werden diese Proben in Agarose eingebettet und in einer Glaskapillare angeordnet. Zur Untersuchung der Probe wird die Glaskapillare in eine wassergefüllte Probenkammer eingebracht. Die Agarose mit der Probe wird ein Stück weit aus der Kapillare herausgedrückt und mit einem Lichtblatt beleuchtet. Die in der Probe angeregte und von dieser ausgehende Fluoreszenz wird mit einem Detektionsobjektiv, das senkrecht zum Lichtblatt und damit auch senkrecht zur Lichtblattoptik (=Beleuchtungsoptik, Beleuchtungsobjektiv) steht, auf einen Detektor, insbesondere eine Kamera, abgebildet.The invention relates to a microscope according to the preamble of independent claim 1. One of the main applications of light sheet microscopy is in the imaging of medium-sized samples, for example organisms, with a size of a few 100 μm down to a few millimeters. Typically, these samples are embedded in agarose and placed in a glass capillary. To examine the sample, the glass capillary is placed in a water-filled sample chamber. The agarose with the sample is squeezed out of the capillary a little and illuminated with a light sheet. The fluorescence excited in the sample and emanating from it is imaged onto a detector, in particular a camera, with a detection objective which is perpendicular to the light sheet and thus also perpendicular to the light sheet optics (=illumination optics, illumination objective).
Ein Aufbau eines Mikroskops 0 für die Lichtblattmikroskopie (SPIM-Aufbau; Single Plane Illumination Microscopy) umfasst gemäß dem Stand der Technik ein Beleuchtungsobjektiv 2 mit einer ersten optischen Achse A1 und ein Detektionsobjektiv 3 mit einer zweiten optischen Achse A2 (auch als SPIM-Objektive bezeichnet), die jeweils unter einem Winkel α1 beziehungsweise a2 von 45° zu einer Probenebene 4 und einem rechten Winkel zueinander von oben auf die Probenebene 4 gerichtet sind (siehe
Die
Dieser Ansatz bietet den Vorteil einer hohen Auflösung in axialer Richtung, da mittels des Beleuchtungsobjektivs 2 ein dünnes Lichtblatt 6 (siehe auch
Probenhalter 7, wie z. B. Multiwellplatten, Petrischalen und/oder Objektträger genutzt und eine Kontamination der Proben 5, insbesondere beim High-Throughput-Screening, vermieden werden.
Weitere technische Schwierigkeiten treten auf, wenn in dem Strahlengang des Beleuchtungsobjektivs 2 und/oder des Detektionsobjektivs 3 beispielsweise Alvarezplatten eines Alvarez-Manipulators 12 (
Aus der
Es gibt mehrere Möglichkeiten, diese Aberrationen zu korrigieren. So ist in der
Gemäß der
Eine weitere Möglichkeit zur Korrektur von Aberrationen eines Mikroskops, die durch ein Deckglas bedingt sind, ist aus der Publikation
Der Erfindung liegt die Aufgabe zugrunde, gegenüber dem Stand der Technik verbesserte Möglichkeiten zur Korrektur von Aberrationen vorzuschlagen, die insbesondere aufgrund schräger Durchgänge von Beleuchtungsstrahlung und Detektionsstrahlung durch optisch brechende Schichten auftreten. Insbesondere soll verbesserte Mikroskope, und hier wiederum insbesondere Lichtblattmikroskope, vorgeschlagen werden.The invention is based on the object of proposing improved options for correcting aberrations compared to the prior art, which in particular are due to oblique passages of illumination radiation and detection radiation through optically refracting layers appear. In particular, improved microscopes, and here in particular light sheet microscopes, are to be proposed.
Die Aufgabe wird durch ein Mikroskop gemäß dem Anspruch 1 gelöst. Vorteilhafte Ausführungen und Weiterbildungen sind Gegenstand der abhängigen Ansprüche.The object is solved by a microscope according to claim 1. Advantageous designs and developments are the subject matter of the dependent claims.
Die Aufgabe wird mittels eines Mikroskops gelöst. Das Mikroskop umfasst eine Beleuchtungsoptik mit einem Beleuchtungsobjektiv zur Beleuchtung einer auf einem Probenträger in einem Probenbereich befindlichen Probe über einen Beleuchtungsstrahlengang, wobei die optische Achse des Beleuchtungsobjektivs in einer Ebene liegt, die mit der Normalen einer Bezugsebene, hinsichtlich welcher der Probenträger ausgerichtet ist, einen von Null verschiedenen Winkel (Beleuchtungswinkel) einschließt. Die Beleuchtung mittels des Beleuchtungsobjektivs erfolgt in der genannten Ebene. Weiterhin ist eine Detektionsoptik mit einem Detektionsobjektiv in einem Detektionsstrahlengang vorhanden. Die optische Achse des Detektionsobjektivs schließt mit der Normalen der Bezugsebene einen von Null verschiedenen Winkel (Detektionswinkel) ein. Das Detektionsobjektiv umfasst ein im Strahlengang angeordnetes oder in diesen einbringbares Detektionskorrekturelement und/oder das Beleuchtungsobjektiv umfasst ein im Strahlengang angeordnetes oder in diesen einbringbares Beleuchtungskorrekturelement.The task is solved using a microscope. The microscope comprises illumination optics with an illumination objective for illuminating a sample located on a sample carrier in a sample area via an illumination beam path, the optical axis of the illumination objective lying in a plane that is one of Includes zero different angles (illumination angles). The illumination by means of the illumination lens takes place in said plane. Furthermore, there is a detection optics with a detection objective in a detection beam path. The optical axis of the detection lens encloses a non-zero angle (detection angle) with the normal of the reference plane. The detection objective includes a detection correction element that is arranged in the beam path or can be introduced into it and/or the illumination objective includes an illumination correction element that is arranged in the beam path or can be introduced into this.
Erfindungsgemäß ist zwischen dem Probenträger und den beiden Objektiven eine Meniskuslinse vorhanden, die sowohl in dem Beleuchtungsstrahlengang als auch in dem Detektionsstrahlengang angeordnet ist. Die Meniskuslinse ist zur Korrektur von Aberrationen ausgebildet, die aufgrund des Durchtritts von zu detektierender Strahlung, insbesondere von Licht, beziehungsweise von Strahlung zur Beleuchtung der Probe durch Medien unterschiedlicher Brechkraft entstehen. Das Korrekturelement beziehungsweise die Korrekturelemente ist beziehungsweise sind zur Korrektur von verbliebenen Aberrationen ausgebildet.According to the invention, a meniscus lens is present between the sample carrier and the two objectives, which is arranged both in the illumination beam path and in the detection beam path. The meniscus lens is designed to correct aberrations that occur due to the passage of radiation to be detected, in particular light, or radiation for illuminating the sample through media of different refractive power. The correction element or the correction elements is or are designed to correct remaining aberrations.
In der Probenebene, die auch als Bezugsebene bezeichnet wird, ist die Probe in einem dafür vorgesehenen Bereich, den Probenbereich, angeordnet oder kann dort angeordnet werden.In the sample plane, which is also referred to as the reference plane, the sample is or can be arranged in a designated area, the sample area.
Die Beleuchtung erfolgt flächig. Zur Vereinfachung der Beschreibung wird im Folgenden auch von einem Korrekturelement beziehungsweise von Korrekturelementen gesprochen, wenn sich die Beschreibung sowohl auf ein Beleuchtungskorrekturelement als auch auf ein Detektionskorrekturelement, oder auf beide bezieht.The lighting is flat. To simplify the description, reference is also made below to a correction element or correction elements if the description relates both to an illumination correction element and to a detection correction element, or to both.
Verbliebene Aberrationen können solche (Rest-)Abbildungsfehler sein, die aus einer unvollständigen Korrektur der Aberrationen aufgrund des schrägen Durchgangs der Strahlungen, seien es Beleuchtungsstrahlung und/oder Detektionsstrahlung, resultieren. Ferner sind verbliebene Aberrationen Fehler beispielsweise aufgrund einer Varianz der Deckglasdicke, Temperaturänderungen, Brechzahlunterschiede durchstrahlter Schichten, Verkippungen des Deckglases oder Keilfehler des Deckglases beruhen. Diese verbliebenen Aberrationen werden korrigiert oder zumindest reduziert. Einem Deckglas sind beispielsweise der Boden eines Probengefäßes oder ein Objektträger aus einem anderen Material als Glas gleichzusetzen.Remaining aberrations can be those (residual) aberrations that result from an incomplete correction of the aberrations due to the oblique passage of the radiation, be it illumination radiation and/or detection radiation. Furthermore, remaining aberrations are errors, for example due to a variance in the cover glass thickness, temperature changes, differences in the refractive index of layers penetrated by radiation, tilting of the cover glass or wedge errors in the cover glass. These remaining aberrations are corrected or at least reduced. For example, the bottom of a sample vessel or a slide made of a material other than glass can be equated with a cover glass.
Das Mikroskop kann ein Trennschichtsystem mit mindestens einer Schicht aus einem vorgegebenen Material mit vorgegebener Dicke aufweisen. Die mindestens eine Schicht, beispielsweise ein Deckglas, trennt ein Medium, in welchem sich die Probe befindet, von dem Beleuchtungsobjektiv und dem Detektionsobjektiv. Das Trennschichtsystem steht mit einer parallel zur Bezugsebene ausgerichteten Grundfläche zumindest in dem für das Beleuchtungsobjektiv und das Detektionsobjektiv für Beleuchtung beziehungsweise Detektion zugänglichen Bereich mit dem Medium und/oder mit einem Immersionsmedium in Kontakt. Das Medium und das Immersionsmedium sind durch das Trennschichtsystem voneinander separiert.The microscope can have a separating layer system with at least one layer made of a specified material with a specified thickness. The at least one layer, for example a cover glass, separates a medium in which the sample is located from the illumination objective and the detection objective. The separating layer system stands, with a base surface aligned parallel to the reference plane, at least in the area for the illumination lens and the Detection objective for illumination or detection accessible area with the medium and / or with an immersion medium in contact. The medium and the immersion medium are separated from each other by the separating layer system.
Die Aberrationen und die verbliebenen Aberrationen sind für einen vorgegebenen Bereich von Beleuchtungswinkeln bzw. von Detektionswinkeln und/oder für einen vorgegebenen Bereich der Dicke der mindestens einen Schicht des Trennschichtsystems reduzierbar.The aberrations and the remaining aberrations can be reduced for a predetermined range of illumination angles or detection angles and/or for a predetermined range of the thickness of the at least one layer of the separating layer system.
Eine Meniskuslinse ist eine Linse, die zwei nach derselben Seite gekrümmte Linsenflächen besitzt. Vorteilhaft weisen die beiden Linsenflächen denselben Mittelpunkt auf. Die beiden Linsenflächen der Meniskuslinse können sich in unterschiedlichen Medien, beispielsweise Immersionsmedien und/oder Luft, mit jeweils unterschiedlichem Brechungsindex befinden. Die Meniskuslinse hat gegenüber dem aus dem Stand der Technik bekannten Virtuellen Relay und gegenüber ebenfalls vorbekannten Freiform-Korrekturlinsen den Vorteil, dass sie einfacher und kostengünstiger herzustellen ist, da keine Freiformflächen aufwendig erzeugt werden müssen.A meniscus lens is a lens that has two lens surfaces curved in the same direction. The two lens surfaces advantageously have the same center point. The two lens surfaces of the meniscus lens can be in different media, for example immersion media and/or air, each with a different refractive index. The meniscus lens has the advantage over the virtual relay known from the prior art and over previously known free-form correction lenses that it can be produced more simply and cost-effectively, since no free-form surfaces have to be produced in a complex manner.
Die Meniskuslinse kann ortsfest gehaltert sein. Die Fokussierung erfolgt durch Verschieben der Probe samt Probenträger oder durch Verschieben der Objektive entlang deren optischer Achse.The meniscus lens can be held stationary. The focusing is done by moving the sample together with the sample carrier or by moving the lenses along their optical axis.
Mittels der Meniskuslinse sind Fehler korrigiert oder korrigierbar, die beim Übergang der Beleuchtungsstrahlung und/oder Detektionsstrahlung zwischen zwei Medien oder Schichten unterschiedlicher Brechzahl auftreten. Aberrationen aufgrund des schiefen Durchgangs werden hingegen nicht korrigiert. Diese verbleibenden Aberrationen können außerhalb und/oder innerhalb des Objektivs mittels des Beleuchtungskorrekturelements und/oder mittels des Detektionskorrekturelements (Korrekturelemente) korrigiert werden.The meniscus lens is used to correct or correct errors that occur when the illumination radiation and/or detection radiation transitions between two media or layers of different refractive index. However, aberrations due to the oblique passage are not corrected. These remaining aberrations can be corrected outside and/or inside the lens by means of the illumination correction element and/or by means of the detection correction element (correction elements).
Um ein erfindungsgemäßes Lichtblattmikroskop zu realisieren, ist eine zur Beleuchtung verwendete Strahlung zu einem Lichtblatt geformt und in den Probenbereich gerichtet. In alternativen Ausführungen wird das Lichtblatt mittels der Beleuchtungsstrahlung in dem Probenbereich erzeugt, indem beispielsweise ein Strahlenbündel der Beleuchtungsstrahlung in der Ebene bewegt wird (dynamisches Lichtblatt). Dabei liegen die optische Achse des Beleuchtungsobjektivs und das Lichtblatt in einer Ebene, die mit der Normalen der Bezugsebene einen von Null verschiedenen Beleuchtungswinkel einschließt.In order to implement a light sheet microscope according to the invention, a radiation used for illumination is formed into a light sheet and directed into the sample area. In alternative embodiments, the light sheet is generated by means of the illumination radiation in the sample area, for example by moving a bundle of rays of the illumination radiation in the plane (dynamic light sheet). The optical axis of the illumination lens and the light sheet lie in a plane that encloses an illumination angle other than zero with the normal of the reference plane.
In einer Ausführung des Mikroskops ist das optische Korrekturelement in einer Pupille des Detektionsobjektivs und/oder des Beleuchtungsobjektivs angeordnet. Beleuchtungsobjektiv und Detektionsobjektiv werden im Folgenden auch vereinfachend als Objektive bezeichnet.In one embodiment of the microscope, the optical correction element is arranged in a pupil of the detection objective and/or the illumination objective. Illumination lens and detection lens are also referred to below as lenses for the sake of simplicity.
Eine Anordnung des optischen Korrekturelements in einer Pupille oder möglichst nahe der Pupille vermeidet vorteilhaft unerwünschte feldabhängige Effekte. In der Pupille wirkt das Korrekturelement für alle Feldpunkte gleich. Je größer eine Distanz zwischen der Pupille und dem jeweiligen optischen Korrekturelement ist, desto mehr Feldabhängigkeiten kommen zum Tragen, sodass die unerwünschten feldabhängigen Effekte mit zunehmender Distanz stärker ausgeprägt sind.An arrangement of the optical correction element in a pupil or as close as possible to the pupil advantageously avoids undesired field-dependent effects. In the pupil, the correction element has the same effect for all field points. The greater the distance between the pupil and the respective optical correction element, the more field dependencies come into play, so that the undesired field-dependent effects are more pronounced as the distance increases.
In einer möglichen Ausführung des erfindungsgemäßen Mikroskops ist das optische Korrekturelement nahe der Pupille angeordnet, wenn sich dieses innerhalb der Schärfentiefe beispielsweise der Tubuslinse des Detektionsobjektivs beziehungsweise des Beleuchtungsobjektivs befindet.In one possible embodiment of the microscope according to the invention, the optical correction element is arranged close to the pupil if it is within the depth of focus, for example, of the tube lens of the detection objective or of the illumination objective.
Sowohl das optische Beleuchtungskorrekturelements als auch das optische Detektionskorrekturelement kann beziehungsweise können als statische Korrekturelemente oder als adaptive Korrekturelemente ausgebildet sein.Both the optical illumination correction element and the optical detection correction element can be embodied as static correction elements or as adaptive correction elements.
Statische Korrekturelemente sind beispielsweise mindestens eine Phasenplatte oder eine Freiformlinse. Die Freiformlinse muss nicht zwingend in der Pupille platziert sein und kann beispielsweise die Frontlinse des jeweiligen Objektivs sein. Ein statisches Korrekturelement wie die Phasenplatte bewirkt eine Korrektur statischer Anteile der Aberrationen.Static correction elements are, for example, at least one phase plate or one free-form lens. The free-form lens does not necessarily have to be placed in the pupil and can be the front lens of the respective objective, for example. A static correction element such as the phase plate corrects static components of the aberrations.
Um verbundene Restaberrationen des Aufbaus sowie probeninduzierte Aberrationen auszugleichen, lassen sich adaptive Korrekturelemente im Beleuchtungs- und Detektionsstrahlengang der Anordnung unterbringen. Dynamische oder variable Anteile der Aberrationen können mittels wenigstens eines adaptiven Korrekturelements korrigiert sein, wobei das adaptive Korrekturelement innerhalb oder außerhalb des Objektivs beziehungsweise der Objektive angeordnet ist beziehungsweise angeordnet sind. Ein adaptives Korrekturelement ist hinsichtlich seiner Korrekturwirkung einstellbar und adaptiv ausgebildet. Somit sind vorteilhaft dynamische Korrekturen der Aberrationen, insbesondere der verbliebenen Aberrationen ermöglicht.In order to compensate for associated residual aberrations of the structure and sample-induced aberrations, adaptive correction elements can be accommodated in the illumination and detection beam path of the arrangement. Dynamic or variable portions of the aberrations can be corrected by means of at least one adaptive correction element, with the adaptive correction element being or being arranged inside or outside the lens or lenses. An adaptive correction element is adjustable and adaptive in terms of its corrective effect. Dynamic corrections of the aberrations, in particular the remaining aberrations, are thus advantageously made possible.
Statische und adaptive Korrekturelemente können in einem erfindungsgemäßen Mikroskop kombiniert sein. So ist in einer möglichen Ausführung der Erfindung das statische Korrekturelement eine Phasenplatte zur Korrektur statischer Aberrationen und im Beleuchtungs- und/oder im Detektionsstrahlengang ist ein adaptives Korrekturelement angeordnet.Static and adaptive correction elements can be combined in a microscope according to the invention. In one possible embodiment of the invention, the static correction element is a phase plate for correcting static aberrations, and an adaptive correction element is arranged in the illumination and/or detection beam path.
Ist jedem Objektiv ein adaptives Korrekturelement zugeordnet oder weist jedes der Objektive ein adaptives Korrekturelement auf, kann eines der adaptiven Korrekturelemente innerhalb und das andere adaptive Korrekturelement außerhalb des jeweiligen Objektivs vorhanden sein.If an adaptive correction element is assigned to each objective or if each of the objectives has an adaptive correction element, one of the adaptive correction elements can be present inside and the other adaptive correction element can be present outside of the respective objective.
Zweckmäßigerweise ist ein adaptives Korrekturelement in einer Pupillenebene des Mikroskops so angeordnet, dass die wirksame Öffnung des adaptiven Korrekturelements und die Größe des veränderlichen adaptiven Korrekturelements oder der veränderlichen adaptiven Korrekturelemente gut zur Größe der Pupille in der Pupillenebene passen und gewünschte Wellenfrontdeformationen für die zu kompensierenden Aberrationen eingestellt werden können und/oder einen erforderlichen Phasenhub für die zu kompensierenden Aberrationen zu liefern. Beispielsweise erreicht man diese Anpassung sowie die Zugänglichkeit des adaptiven Korrekturelements durch eine Pupillenrelay-Optik, welche die Objektivpupille des Beleuchtungs- oder Detektionsobjektives auf das adaptive Korrekturelement abbildet. Ausreichend kleine adaptive Korrekturelemente lassen sich auch direkt im oder unmittelbar nach dem Objektiv anordnen.An adaptive correction element is expediently arranged in a pupil plane of the microscope in such a way that the effective opening of the adaptive correction element and the size of the variable adaptive correction element or the variable adaptive correction elements fit well with the size of the pupil in the pupil plane and desired wavefront deformations are set for the aberrations to be compensated and/or to supply a required phase shift for the aberrations to be compensated. For example, this adaptation and the accessibility of the adaptive correction element are achieved by a pupil relay optics, which images the objective pupil of the illumination or detection objective onto the adaptive correction element. Sufficiently small adaptive correction elements can also be arranged directly in or immediately after the lens.
Adaptive Korrekturelemente sind beispielsweise adaptive Spiegel oder mindestens ein räumlicher Modulator für Licht (SLM, spatial light modulator). Der SLM kann als ein reflektierender SLM oder als ein transmissiver SLM ausgebildet sein.Adaptive correction elements are, for example, adaptive mirrors or at least one spatial light modulator (SLM). The SLM can be designed as a reflective SLM or as a transmissive SLM.
Das adaptive Korrekturelement ist in weiteren Ausführungen ein Alvarez-Manipulator, mindestens ein adaptiver Spiegel, mindestens eine Kipplinse, mindestens eine Schiebelinse, mindestens eine verformbare optische Linse oder eine Kombination daraus.In further embodiments, the adaptive correction element is an Alvarez manipulator, at least one adaptive mirror, at least one tilting lens, at least one shifting lens, at least one deformable optical lens or a combination thereof.
In einer weiteren Ausführung der Erfindung ist das adaptive Korrekturelement ein räumlicher Modulator für Licht während im Strahlengang eines der Objektive, insbesondere imIn a further embodiment of the invention, the adaptive correction element is a spatial modulator for light while in the beam path of one of the lenses, in particular in the
Detektionsstrahlengang, eine Zylinderlinse zur Teilkompensation von auftretenden Aberrationen vorhanden ist.Detection beam path, a cylindrical lens for partial compensation of aberrations that occur is present.
Es ist ferner möglich, dass das adaptive Korrekturelement ein adaptiver Spiegel ist und im Strahlengang eines der Objektive, insbesondere im Detektionsstrahlengang, eine Zylinderlinse zur Teilkompensation von auftretenden Astigmatismus vorhanden ist.It is also possible for the adaptive correction element to be an adaptive mirror and for a cylindrical lens to be present in the beam path of one of the lenses, in particular in the detection beam path, for partial compensation of astigmatism that occurs.
In einer Ausführung mit einem adaptiven Spiegel wird mittels eines Teleskops die Pupille des Objektivs, sei es das Beleuchtungsobjektiv oder das Detektionsobjektiv, auf den adaptiven Spiegel abgebildet. Der adaptive Spiegel wird so verformt, dass durch diesen die auftretenden Aberrationen korrigiert und reduziert werden. Ein nahezu oder gänzlich aberrationsfreies Bild kann mittels einer weiteren im Detektionsstrahlengang angeordneten optischen Linse auf dem Kamerasensor erzeugt werden.In an embodiment with an adaptive mirror, the pupil of the lens, be it the illumination lens or the detection lens, is imaged onto the adaptive mirror by means of a telescope. The adaptive mirror is deformed in such a way that it corrects and reduces the aberrations that occur. An almost or completely aberration-free image can be generated on the camera sensor by means of a further optical lens arranged in the detection beam path.
Ist das adaptive Korrekturelement durch einen reflektierenden SLM realisiert, wird mittels eines Teleskops die Pupille des Objektivs auf den SLM abgebildet. Auf dem SLM wird ein Phasenmuster angezeigt, durch dessen Wirkung die auftretenden Aberrationen korrigiert und reduziert werden. Wiederum kann ein nahezu oder gänzlich aberrationsfreies Bild mittels einer weiteren im Detektionsstrahlengang angeordneten optischen Linse auf dem Kamerasensor erzeugt werden.If the adaptive correction element is implemented by a reflecting SLM, the pupil of the lens is imaged onto the SLM using a telescope. A phase pattern is displayed on the SLM, the effect of which is to correct and reduce the aberrations that occur. Again, an almost or completely aberration-free image can be generated on the camera sensor by means of a further optical lens arranged in the detection beam path.
Der SLM ist in einer weiteren Ausführung mit einer Zylinderlinse kombiniert. Die Zylinderlinse wird z. B. in der Pupille des Objektivs verwendet, um eine Teilkorrektur der auftretenden Aberrationen durchzuführen. Mittels eines Teleskops wird die Pupille des Objektivs auf einen reflektierenden SLM abgebildet. Auf dem SLM wird ein Phasenmuster angezeigt, durch dessen optische Wirkung die verbleibenden Restaberrationen korrigiert und reduziert werden . Durch eine dritte Linse wird ein nahezu oder gänzlich aberrationsfreies Bild auf dem Kamerasensor erzeugt.In another version, the SLM is combined with a cylindrical lens. The cylindrical lens is z. B. used in the pupil of the lens to perform a partial correction of the aberrations that occur. The pupil of the lens is imaged onto a reflecting SLM using a telescope. A phase pattern is displayed on the SLM, the optical effect of which corrects and reduces the remaining residual aberrations. A third lens creates an almost or completely aberration-free image on the camera sensor.
Die adaptiven Korrekturelemente können im Beleuchtungs- und/oder im Detektionsstrahlengang angeordnet sein.The adaptive correction elements can be arranged in the illumination and/or in the detection beam path.
Des Weiteren können die Aberrationen auch innerhalb des Objektivs korrigiert werden.Furthermore, the aberrations can also be corrected within the lens.
Dazu wird zum Beispiel im Objektiv eine zusätzliche Pupille geschaffen, an deren Stelle das adaptive Korrekturelement platziert wird, um die auftretenden Aberrationen zu korrigieren. Auch hier kann eine Zylinderlinse verwendet werden, um eine Teilkorrektur der Aberrationen durchzuführen.For this purpose, an additional pupil is created in the lens, for example, and the adaptive correction element is placed in its place in order to correct the aberrations that occur. A cylindrical lens can also be used here in order to carry out a partial correction of the aberrations.
Die Erfindung ist insbesondere in Form eines inversen Lichtblattmikroskops mit schrägem Durchgang der Beleuchtungs- und Detektionsstrahlung durch eine Probenhalterung beispielsweise in Form eines Deckglases oder einer optisch transparenten Schicht wie dem Boden einer Petrischale geeignet.The invention is particularly suitable in the form of an inverted light sheet microscope with oblique passage of the illumination and detection radiation through a sample holder, for example in the form of a cover glass or an optically transparent layer such as the bottom of a Petri dish.
Die Erfindung wird nachfolgend anhand von Ausführungsbeispielen und Abbildungen näher erläutert. Dabei zeigen:
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Fig. 1a eine schematische Darstellung einer Anordnung eines Lichtblattmikroskops gemäß dem Stand der Technik, -
Fig. 1b eine schematische Darstellung einer inversen Anordnung eines Lichtblattmikroskops gemäß dem Stand der Technik, -
Fig. 2 eine schematische Darstellung eines ersten Ausführungsbeispiels einer erfindungsgemäßen Anordnung eines Lichtblattmikroskops, -
Fig. 3 eine schematische Darstellung eines zweiten Ausführungsbeispiels einer erfindungsgemäßen Anordnung eines Lichtblattmikroskops, -
Fig. 4 eine schematische Darstellung eines dritten Ausführungsbeispiels einer erfindungsgemäßen Anordnung eines Lichtblattmikroskops, -
Fig. 5 eine schematische Darstellung eines vierten Ausführungsbeispiels einer erfindungsgemäßen Anordnung eines Lichtblattmikroskops und -
Fig. 6 eine schematische Darstellung eines Ausführungsbeispiels eines Objektivs zur Verwendung in einem der erfindungsgemäßen Anordnungen eines Mikroskops.
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Fig. 1a a schematic representation of an arrangement of a light sheet microscope according to the prior art, -
Fig. 1b a schematic representation of an inverse arrangement of a light sheet microscope according to the prior art, -
2 a schematic representation of a first exemplary embodiment of an arrangement according to the invention of a light sheet microscope, -
3 a schematic representation of a second exemplary embodiment of an arrangement according to the invention of a light sheet microscope, -
4 a schematic representation of a third exemplary embodiment of an arrangement according to the invention of a light sheet microscope, -
figure 5 a schematic representation of a fourth exemplary embodiment of an arrangement according to the invention of a light sheet microscope and -
6 a schematic representation of an embodiment of a lens for use in one of the inventive arrangements of a microscope.
Die
Als wesentliche Elemente einer erfindungsgemäßen Anordnung zur Mikroskopie, insbesondere zur Lichtblattmikroskopie, sind neben dem schräg zur Proben- oder Bezugsebene 4 ausgerichteten Beleuchtungsobjektiv 2 und dem ebenfalls schräg zur Bezugsebene 4 ausgerichteten Detektionsobjektiv 3 eine gemeinsam genutzte Meniskuslinse 10 (
Die nachfolgenden Ausführungsbeispiele sind beispielhaft anhand inverser Mikroskope 0 dargestellt und können in weiteren Ausführungen auch als aufrechte Mikroskope 0 ausgebildet sein.The following exemplary embodiments are shown as examples using
Ein Ausführungsbeispiel eines zur Lichtblattmikroskopie ausgebildeten Anordnung 1 eines inversen Mikroskops 0 (nur symbolisch dargestellt) mit Korrekturelementen 2KE, 3KE und einer Meniskuslinse 10 ist in
Die Probenhalterung 7 ist auf dem Probentisch 11 gehalten. Der Probentisch 11 selbst ist in einer durch die X-Achse X und die Y-Achse Y aufgespannten X-Y-Ebene mittels nicht näher dargestellter Antriebe gesteuert verstellbar.The
Das Beleuchtungsobjektiv 2 und das Detektionsobjektiv 3 sind jeweils mittels eines Objektivantriebs 14, der hier als ein Piezoantrieb ausgebildet ist, entlang der ersten optischen Achse A1 beziehungsweise entlang der zweiten optischen Achse A2 gesteuert verstellbar.The
Die Beleuchtungsstrahlung BS wird durch ein Lasermodul 15 bereitgestellt und mittels einer Strahlformungseinheit 16 geformt. Die Strahlformungseinheit 16 ist beispielsweise eine Optik, mittels der die bereitgestellte Beleuchtungsstrahlung BS geformt, beispielsweise kollimiert, wird.The illumination radiation BS is provided by a
Der Strahlformungseinheit 16 nachgeordnet ist ein Scanner 17 vorhanden, mittels dem die geformte Beleuchtungsstrahlung BS gesteuert in zwei Richtungen ablenkbar ist (X-Y-Scanner).Downstream of the
Nach dem Scanner 17 ist das Beleuchtungsobjektiv 2 auf der ersten optischen Achse A1 angeordnet. Die von dem Scanner 17 abgelenkte Beleuchtungsstrahlung BS gelangt zu dem Beleuchtungsobjektiv 2 und wird durch dieses geformt und/oder fokussiert. Das Lichtblatt 6 wird durch eine entsprechende Ablenkung der Beleuchtungsstrahlung BS mittels des Scanners 17 in einem Probenbereich erzeugt, in dem sich die Probe 5 befindet.After the
Die von der Probe 5 und aus dem Probenbereich kommende Detektionsstrahlung DS ist entlang der zweiten optischen Achse A2 auf einen Detektor 19 gerichtet und durch diesen erfassbar.The detection radiation DS coming from the
Zur Ansteuerung des Probentischs 11, der Objektivantriebe 14, der Korrekturelemente 2KE, 3KE, des Lasermoduls 15, der Strahlformung 16, des Scanners 17 und/oder des Detektors 19 ist eine Steuereinheit 13 vorhanden, die mit den anzusteuernden Elementen in einer zur Datenübertragung geeigneten Verbindung steht (nur angedeutet gezeigt).A
In weiteren Ausführungen ist die Steuereinheit 13 zusätzlich zur Erfassung, Speicherung und/oder zur Auswertung von Messwerten konfiguriert. Mittels der Steuereinheit 13 können weitere Elemente sowie Einheiten des Mikroskops 0 ansteuerbar sein und/oder Messwerte von diesen erhalten und ausgewertet werden.In further versions, the
Es werden im Folgenden zur Beschreibung zwei Koordinatensysteme mit zueinander orthogonalen Achsen genutzt. Das erste Koordinatensystem ist das Koordinatensystem der gesamten Anordnung mit einer X-Achse X, einer Y-Achse Y und einer Z-Achse Z. Idealerweise ist die Probenhalterung 7, insbesondere deren Boden, parallel zu einer durch die X-Achse X und die Y-Achse Y aufgespannte X-Y-Ebene ausgerichtet. Das zweite Koordinatensystem ist das Koordinatensystem des Detektors 19 mit der X-Achse X, einer y-Achse y' und einer z-Achse z'. Eine Abbildung beispielsweise eines Bildes aus der Bildebene BE auf dem Detektor 19 besitzt die Koordinaten X und y'. Die X-Achse X ist in beiden Koordinatensystemen orthogonal zur Zeichenebene der Figuren gerichtet. Die beiden anderen Achsen Y und y' beziehungsweise Z und z', können durch eine Rotation um die X-Achse X ineinander überführt werden.In the following, two coordinate systems with mutually orthogonal axes are used for the description. The first coordinate system is the coordinate system of the entire arrangement with an X-axis X, a Y-axis Y and a Z-axis Z. Ideally, the
Aberrationen, die bei einem schiefen Durchgang der Beleuchtungsstrahlung BS durch die Probenhalterung 7 auftreten, sind von deren Dicke abhängig. Aus diesem Grund sind beispielsweise die Korrekturelemente 2KE, 3KE verschiebbar in den Beleuchtungsobjektiv 2 und/oder dem Detektionsobjektiv 3 gelagert, um durch eine Verschiebung der Korrekturelemente 12 zueinander eine Aberrationskorrektur auf die Dicke abzustimmen.Aberrations that occur when the illumination radiation BS passes through the
Der Boden der Probenhalterung 7 stellt ein Trennschichtsystem mit mindestens einer Schicht aus einem vorgegebenen Material mit vorgegebener Dicke dar, welche ein Medium 8, in dem sich die Probe 5 befindet, von dem Beleuchtungsobjektiv 2 und dem Detektionsobjektiv 3 trennt. Das Trennschichtsystem steht mit einer parallel zur Probenebene 4 ausgerichteten Grundfläche zumindest in dem für das Beleuchtungsobjektiv 2 und das Detektionsobjektiv 3 für die Beleuchtung beziehungsweise die Detektion zugänglichen Bereich mit dem Immersionsmedium 18 in Kontakt.The bottom of the
In einem in der
Beleuchtungskorrekturelemente 2KE auf. Im Strahlengang der Detektionsstrahlung DS sind optische Linsen 20 vorhanden, mittels denen die Detektionsstrahlung DS auf einLighting correction elements 2KE on.
Detektionskorrekturelement 3KE in Form eines SLM oder eines adaptiven Spiegels gelenkt wird. Die von dem Detektionskorrekturelement 3KE reflektierte Detektionsstrahlung DS wird auf einen Detektor 19 gerichtet und durch diesen erfasst.Detection correction element 3KE is directed in the form of an SLM or an adaptive mirror. The detection radiation DS reflected by the detection correction element 3KE is directed onto a
In einer weiteren Ausführungsmöglichkeit ist der SLM als ein transmissiver SLM ausgebildet.In a further possible embodiment, the SLM is designed as a transmissive SLM.
Die
Ein weiteres Ausführungsbeispiel eines erfindungsgemäßen Mikroskops (0) ist in der
Ein Ausführungsbeispiel eines Objektivs 2,3 zur Verwendung in einem erfindungsgemäßen Mikroskop 0 (siehe
Neben den lediglich beispielhaft gezeigten optischen Linsen 20 ist in dem Strahlengang ein als ein SLM ausgebildetes ansteuerbares Korrekturelement 2KE beziehungsweise 3KE angeordnet. Die Ansteuerung erfolgt mittels der Steuereinheit 13.In addition to the
In weiteren Ausführungen des Mikroskops (0) oder des Objektivs 2, 3 können Schiebelinsen vorgesehen sein, die radial zum Strahlengang verschiebbar sind um verbliebene Aberrationen auszugleichen oder zumindest zu reduzieren.In further versions of the microscope (0) or the
In dem in
- 00
- Mikroskopmicroscope
- 11
- Anordnungarrangement
- 22
- Beleuchtungsobjektivlighting lens
- 2KE2KE
- Beleuchtungskorrekturelementlighting correction element
- 33
- Detektionsobjektivdetection lens
- 3KE3KE
- Detektionskorrekturelementdetection correction element
- 44
- Probenebene (= Bezugsebene)sample plane (= reference plane)
- 55
- Probesample
- 66
- Lichtblattlight sheet
- 77
- Probenhalterungsample holder
- 88th
- Mediummedium
- 99
- Zylinderlinsecylindrical lens
- 1010
- Meniskuslinsemeniscus lens
- 1111
- Probentischrehearsal table
- 1212
- Alvarez-ManipulatorAlvarez manipulator
- 1313
- Steuereinheitcontrol unit
- 1414
- Objektivantrieblens drive
- 1515
- Lasermodullaser module
- 1616
- Strahlformungbeam shaping
- 1717
- X-Y-ScannerX-Y scanner
- 1818
- Immersionsmediumimmersion medium
- 1919
- Detektordetector
- 2020
- optische Linseoptical lens
- A1A1
- erste optische Achse (optische Achse des Beleuchtungsobjektivs 2)first optical axis (optical axis of illumination lens 2)
- A2A2
- zweite optische Achse (optische Achse des Detektionsobjektivs 3)second optical axis (optical axis of detection lens 3)
- α1α1
- Winkel / Beleuchtungswinkelangle / lighting angle
- a2a2
- Winkel / DetektionswinkelAngle / detection angle
- BB
- Normalenormal
- BEBE
- Bildebenepicture plane
- BSB.S
- Beleuchtungsstrahlungillumination radiation
- DSDS
- Detektionsstrahlungdetection radiation
Claims (9)
- Microscope (0), comprising- a specimen carrier (7) aligned with respect to a specimen plane (4);- an illumination optical unit with an illumination objective (2) for illuminating, by way of an illumination beam path, a specimen (5) situated on the specimen carrier (7) in a specimen region of the specimen plane (4), wherein- a radiation utilized for illumination purposes is shaped into a light sheet (6) and directed into the specimen region, and the optical axis (A1) of the illumination objective (2) and the light sheet (6) lie in a plane that includes an illumination angle (α1) that differs from zero with the normal of the specimen plane (4), and the illumination is implemented in the plane,- a detection optical unit with a detection objective (3) in a detection beam path, the optical axis (A2) of which includes a detection angle (α2) that differs from zero with the normal of the specimen plane (4) and is perpendicular to the light sheet (6),- the illumination objective (2) comprises an illumination correction element (2KE) that is arranged in the beam path, and/or- the detection objective (3) comprises a detection correction element (3KE) that is arranged in the beam path,characterized in that- a meniscus lens (10) is present between the specimen carrier (7) and the objectives (2, 3), said meniscus lens being arranged both in the illumination beam path and in the detection beam path, wherein the meniscus lens (10) has two lens surfaces curved to the same side and the two lens surfaces have the same centre;- the meniscus lens (10) is embodied to correct aberrations that arise on account of the passage through media with different refractive indices of radiation to be detected and/or radiation for illuminating the specimen, and- the illumination correction element (2KE) and/or the detection correction element (3KE) is embodied to correct remaining aberrations.
- Microscope (0) according to Claim 1, characterized in that the illumination correction element (2KE) and/or the detection correction element (3KE) is arranged in a pupil of the illumination objective (2) and/or of the detection objective (3).
- Microscope (0) according to either of Claims 1 and 2, characterized in that the illumination correction element (2KE) and/or the detection correction element (3KE) is at least one phase plate or a free-form lens.
- Microscope (0) according to either of Claims 1 and 2, characterized in that the illumination correction element (2KE) and/or the detection correction element (3KE) is a phase plate for correcting static aberrations and an adaptive mirror or a spatial light modulator is present in the illumination beam path and/or in the detection beam path.
- Microscope (0) according to either of Claims 1 and 2, characterized in that the illumination correction element (2KE) and/or the detection correction element (3KE) has an adjustable and adaptive embodiment in respect of its corrective power.
- Microscope (0) according to Claim 5, characterized in that the adaptive illumination correction element (2KE) and/or the adaptive detection correction element (3KE) is an Alvarez manipulator (12), a spatial light modulator, at least one adaptive mirror, at least one tilt lens, at least one sliding lens, at least one deformable optical lens or a combination thereof.
- Microscope (0) according to Claim 5, characterized in that the adaptive illumination correction element (2KE) and/or the adaptive detection correction element (3KE) is a spatial light modulator and a cylindrical lens (9) is present in the detection beam path for partial compensation of occurring astigmatism.
- Microscope (0) according to Claim 5, characterized in that the adaptive detection correction element (3KE) is an adaptive mirror and a cylindrical lens (9) is present in the illumination beam path and/or in the detection beam path for partial compensation of occurring aberrations.
- Microscope (0) according to any one of the preceding claims, characterized by a separation layer system with at least one layer made of a predetermined material with a predetermined thickness, which separates a medium (8), in which the specimen (5) is situated, from the illumination objective (2) and the detection objective (3), wherein, by means of a base that is aligned parallel to the specimen plane (4), the separation layer system is in contact with an immersion medium (18), at least in the region accessible to the illumination objective (2) and the detection objective (3) for illumination and detection purposes, respectively.
Applications Claiming Priority (2)
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DE102016212020.4A DE102016212020A1 (en) | 2016-07-01 | 2016-07-01 | Arrangement for microscopy and correction of aberrations |
PCT/EP2017/066136 WO2018002225A1 (en) | 2016-07-01 | 2017-06-29 | Arrangement for microscopy and for correction of aberrations |
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EP3479158A1 EP3479158A1 (en) | 2019-05-08 |
EP3479158B1 true EP3479158B1 (en) | 2022-10-12 |
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US (1) | US11163147B2 (en) |
EP (1) | EP3479158B1 (en) |
JP (2) | JP7308033B2 (en) |
CN (1) | CN109416462B (en) |
DE (2) | DE102016212020A1 (en) |
WO (1) | WO2018002225A1 (en) |
Families Citing this family (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102016212019A1 (en) | 2016-07-01 | 2018-01-04 | Carl Zeiss Microscopy Gmbh | Inclination measurement and correction of the cover glass in the beam path of a microscope |
DE102016120683A1 (en) * | 2016-10-28 | 2018-05-03 | Carl Zeiss Microscopy Gmbh | Light sheet microscope |
DE102017204325A1 (en) * | 2017-03-15 | 2018-09-20 | Carl Zeiss Microscopy Gmbh | Arrangement, microscope and method for TIRF microscopy |
DE102017105928B4 (en) * | 2017-03-20 | 2024-08-14 | Carl Zeiss Microscopy Gmbh | Microscope and method for imaging an object |
DE102017214189A1 (en) * | 2017-08-15 | 2019-02-21 | Carl Zeiss Microscopy Gmbh | Method for operating a microscope assembly and a microscope assembly with a first microscope and at least one further microscope |
DE102017217389A1 (en) | 2017-09-29 | 2019-04-04 | Carl Zeiss Microscopy Gmbh | An optical lens for use in a media delivery device and lens, media delivery device and microscope |
DE102017223014A1 (en) * | 2017-12-18 | 2019-06-19 | Carl Zeiss Microscopy Gmbh | Method for determining the thickness of a sample holder in the beam path of a microscope |
DE102018222839A1 (en) * | 2018-08-10 | 2020-02-13 | Leica Microsystems Cms Gmbh | Illumination arrangement for a microscope, microscope and method for illuminating a sample volume in a microscope |
CN113892042B (en) | 2019-01-14 | 2023-07-14 | 华盛顿大学 | Apparatus, system and method for solid immersion meniscus lenses |
DE102019109832B3 (en) * | 2019-04-12 | 2020-04-23 | Leica Microsystems Cms Gmbh | Light sheet microscope and method for acquiring a measured variable |
DE102019208232A1 (en) * | 2019-06-05 | 2020-12-10 | Carl Zeiss Microscopy Gmbh | Optical arrangement and method for correcting centering errors and / or angle errors |
US20200408691A1 (en) * | 2019-06-27 | 2020-12-31 | Nikita Vladimirov | Multi-view light-sheet microscope with an optical arm combiner |
DE102019214929B4 (en) * | 2019-09-27 | 2022-02-10 | Leica Microsystems Cms Gmbh | Compact light sheet microscope and use of a finitely corrected objective in a light sheet microscope |
EP3805837B1 (en) * | 2019-10-10 | 2024-01-10 | Leica Microsystems CMS GmbH | Microscope system comprising a wave front manipulator element |
EP3907548B1 (en) | 2020-05-04 | 2024-01-10 | Leica Microsystems CMS GmbH | Light sheet microscope and method for imaging an object |
EP3945358B1 (en) | 2020-07-30 | 2024-04-24 | Leica Microsystems CMS GmbH | Oblique plane microscope and method for operating the same |
DE102020211148A1 (en) | 2020-09-04 | 2022-03-10 | Carl Zeiss Microscopy Gmbh | Arrangement for light sheet microscopy, immersion objective and method for reducing aberrations |
DE102020128524A1 (en) | 2020-10-29 | 2022-05-05 | Carl Zeiss Microscopy Gmbh | Light sheet microscope and method of light sheet microscopy |
CN113933273A (en) * | 2021-09-29 | 2022-01-14 | 深圳高性能医疗器械国家研究院有限公司 | Lens, sample bin and imaging system for light sheet fluorescence microscope |
CN113768472B (en) * | 2021-11-10 | 2022-03-22 | 华中科技大学 | Three-dimensional image acquisition device with fluorescent marker and method |
DE102022200841B3 (en) | 2022-01-26 | 2023-05-04 | Carl Zeiss Microscopy Gmbh | Method, arrangement and microscope for three-dimensional imaging in microscopy using an asymmetric point spread function |
US11921586B2 (en) * | 2022-03-03 | 2024-03-05 | Dell Products L.P. | Method and system for backing up virtual machines in a cluster shared volume |
Family Cites Families (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3305294A (en) | 1964-12-03 | 1967-02-21 | Optical Res & Dev Corp | Two-element variable-power spherical lens |
US3497290A (en) * | 1967-07-03 | 1970-02-24 | Bausch & Lomb | 100x magnification semi-objective with field flattening lens |
JP3371934B2 (en) * | 1995-03-07 | 2003-01-27 | 株式会社ニコン | Microscope objective lens |
JPH10142510A (en) * | 1996-11-08 | 1998-05-29 | Nikon Corp | Objective lens for microscope |
JP2000058436A (en) | 1998-08-11 | 2000-02-25 | Nikon Corp | Projection aligner and exposure method |
JP5523840B2 (en) * | 2007-02-09 | 2014-06-18 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | System and method for adjusting spherical aberration of objective lens |
JP2008276070A (en) | 2007-05-02 | 2008-11-13 | Olympus Corp | Magnifying image pickup apparatus |
JP5536995B2 (en) * | 2007-07-17 | 2014-07-02 | オリンパス株式会社 | Microscope objective lens and laser scanning microscope system |
JP2009162974A (en) | 2008-01-07 | 2009-07-23 | Olympus Corp | Microscope system and its control method |
WO2009096522A1 (en) | 2008-02-01 | 2009-08-06 | Nikon Corporation | Microscope and aberration correction controlling device |
DE102010039746B4 (en) | 2010-08-25 | 2016-02-25 | Carl Zeiss Ag | Aberration corrected microscope |
EP2887132B1 (en) * | 2012-08-16 | 2019-10-02 | Citizen Watch Co., Ltd. | Aberration-correcting optical unit and laser microscope |
EP3605183A1 (en) * | 2013-05-10 | 2020-02-05 | European Molecular Biology Laboratory | Microscope for selective plane illumination microscopy |
DE102013107297A1 (en) | 2013-07-10 | 2015-01-15 | Carl Zeiss Microscopy Gmbh | Arrangement for light-sheet microscopy |
DE102013112595B4 (en) * | 2013-11-15 | 2024-08-01 | Carl Zeiss Microscopy Gmbh | Arrangement for light sheet microscopy |
DE102013112600A1 (en) * | 2013-11-15 | 2015-05-21 | Carl Zeiss Microscopy Gmbh | Optical transmission system and microscope with such a transmission system |
DE102013112596B4 (en) * | 2013-11-15 | 2023-12-28 | Carl Zeiss Microscopy Gmbh | Arrangement for light sheet microscopy |
DE102014104977B4 (en) | 2014-04-08 | 2023-11-30 | Carl Zeiss Microscopy Gmbh | Arrangement for light sheet microscopy and microscope objective for light sheet microscopy |
GB2541308B (en) | 2014-05-29 | 2021-05-19 | Hitachi High Tech Corp | Microscope observation container and observation device |
EP3201672A4 (en) * | 2014-10-02 | 2018-05-23 | The Regents of The University of California | Selective plane illumination microscopy (spim) systems and methods |
JP6367685B2 (en) * | 2014-10-24 | 2018-08-01 | オリンパス株式会社 | Microscope objective lens |
DE102014119255A1 (en) * | 2014-12-19 | 2016-06-23 | Carl Zeiss Microscopy Gmbh | Method for light-sheet microscopic examination of a sample |
DE102016120683A1 (en) * | 2016-10-28 | 2018-05-03 | Carl Zeiss Microscopy Gmbh | Light sheet microscope |
DE102017204325A1 (en) * | 2017-03-15 | 2018-09-20 | Carl Zeiss Microscopy Gmbh | Arrangement, microscope and method for TIRF microscopy |
DE102017217389A1 (en) * | 2017-09-29 | 2019-04-04 | Carl Zeiss Microscopy Gmbh | An optical lens for use in a media delivery device and lens, media delivery device and microscope |
-
2016
- 2016-07-01 DE DE102016212020.4A patent/DE102016212020A1/en active Pending
- 2016-07-01 DE DE202016008115.3U patent/DE202016008115U1/en active Active
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WO2018002225A1 (en) | 2018-01-04 |
JP7308033B2 (en) | 2023-07-13 |
DE202016008115U1 (en) | 2017-03-01 |
US20190170995A1 (en) | 2019-06-06 |
EP3479158A1 (en) | 2019-05-08 |
US11163147B2 (en) | 2021-11-02 |
CN109416462A (en) | 2019-03-01 |
JP2019521385A (en) | 2019-07-25 |
JP2022060260A (en) | 2022-04-14 |
DE102016212020A1 (en) | 2018-01-04 |
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